Romsicki Y, Sharom F J
Guelph-Waterloo Centre for Graduate Work in Chemistry and Biochemistry, Department of Chemistry and Biochemistry, University of Guelph, Ontario, Canada.
Eur J Biochem. 1998 Aug 15;256(1):170-8. doi: 10.1046/j.1432-1327.1998.2560170.x.
P-glycoprotein functions as an active efflux pump for lipophilic compounds and plays an important role in the resistance of human cancers to chemotherapeutic drugs. Drug transport is powered by ATP hydrolysis at two highly conserved nucleotide-binding domains, which are proposed to be located at the cytosolic face of the protein. The ATPase activity of P-glycoprotein depends on the presence of phospholipids, and various lipids affect both basal ATPase activity and its stimulation or inhibition by drug substrates. The modulating effects of the lipid-phase state and effects on the function of the nucleotide-binding domains of P-glycoprotein have been studied in reconstituted vesicles of the synthetic phospholipids 1-palmitoyl-2-myristoylphosphatidylcholine (PamMyrGroPCho) and dimyristoylphosphatidylcholine (Myr2GroPCho). The kinetic parameters for P-glycoprotein ATPase activity were determined, and a fluorescence-quenching technique was used to measure the Kd for ATP binding. The values of both the Km for ATP hydrolysis and Kd for ATP binding were significantly different above and below the gel/liquid-crystalline phase transition temperature (tm) of PamMyrGroPCho and Myr2GroPCho, whereas they were similar at the same temperatures for P-glycoprotein in detergent solution. A discontinuity at 21-24 degrees C was observed in the Arrhenius plots of P-glycoprotein ATPase activity in a membrane environment, but not in detergent solution. In addition, the activation energies for ATP hydrolysis in the gel and liquid-crystalline phases of the lipid bilayer were significantly different. P-glycoprotein in PamMyrGroPCho bilayers displayed an unusually low activation energy just below the melting transition. These results indicate that both ATP binding and ATP hydrolysis by P-glycoprotein are affected by the phase state of the host lipids in which it is reconstituted. Lipids may modulate the function of the nucleotide-binding domains of P-glycoprotein by interacting with the transmembrane regions of the protein, or the nucleotide-binding domains themselves may interact with the surface of the bilayer.
P-糖蛋白作为亲脂性化合物的活性外排泵，在人类癌症对化疗药物的耐药性中起重要作用。药物转运由两个高度保守的核苷酸结合结构域处的ATP水解提供动力，这两个结构域被认为位于蛋白质的胞质面。P-糖蛋白的ATP酶活性取决于磷脂的存在，各种脂质既影响基础ATP酶活性，也影响药物底物对其的刺激或抑制作用。在合成磷脂1-棕榈酰-2-肉豆蔻酰磷脂酰胆碱（PamMyrGroPCho）和二肉豆蔻酰磷脂酰胆碱（Myr2GroPCho）的重构囊泡中研究了脂质相状态的调节作用以及对P-糖蛋白核苷酸结合结构域功能的影响。测定了P-糖蛋白ATP酶活性的动力学参数，并使用荧光猝灭技术测量ATP结合的解离常数（Kd）。在PamMyrGroPCho和Myr2GroPCho的凝胶/液晶相转变温度（tm）之上和之下，ATP水解的米氏常数（Km）和ATP结合的Kd值均有显著差异，而在相同温度下，去污剂溶液中的P-糖蛋白的这些值相似。在膜环境中，P-糖蛋白ATP酶活性的阿伦尼乌斯图在21-24摄氏度处出现间断，但在去污剂溶液中未观察到。此外，脂质双层的凝胶相和液晶相中ATP水解的活化能有显著差异。PamMyrGroPCho双层中的P-糖蛋白在刚好低于熔化转变温度时显示出异常低的活化能。这些结果表明，P-糖蛋白的ATP结合和ATP水解均受其重构所在宿主脂质的相状态影响。脂质可能通过与蛋白质的跨膜区域相互作用来调节P-糖蛋白核苷酸结合结构域的功能，或者核苷酸结合结构域本身可能与双层表面相互作用。
